Influenza is a constant and serious threat to public health. Each year, influenza epidemic leads to 200,000 hospitalizations and 36,000 deaths in the United States. Globally, influenza disease impacts every year up to 10% of the world's population—approximately 500 millions of the people.
An influenza pandemic could lead to far greater number of deaths and economic impact. The 1918 influenza pandemic, for example, killed 20-40 million people in the world and more than 500,000 people in the United States. As medical science advances, there are several drugs that are available for treatment or prophylaxis of influenza A and B. However, the prerequisite for effective treatment or prevention control is rapid, sensitive and specific detection of the viruses at an early stage of individual infection or of an outbreak.
Conventional method for influenza virus detection involves first viral culture of a nasal wash, throat swab specimen, tracheal aspirate secretions, bronchial lavages, or lung tissue. The virus in cultures usually is detected between day 2 and day 5 by performing hemabsorption test, which is normally followed by detection with an immunofluorescence assay (IFA) using type specific antibodies. This test, while reliable, is not suitable for point-of-care use, which requires a test that can produce a test result in a short period of time, e.g., less than 30 minutes.
Consequently, faster influenza virus tests were developed to meet the need of point-of-care use. These tests are of two types. One uses specific antibody or antibodies to detect influenza proteins, or antigens. The other detects the activity of neuraminidase that is present in all type A and B influenza virus. Wile these tests can generally be completed in a short period of time, e.g., within 30 minutes, they lack sufficient clinical sensitivity, which is generally around 70% when compared to virus culture methods. Lack of clinical sensitivity for these rapid tests appears to be a result of low analytical sensitivity (or limit of detection), which is generally in the range between 104 and 105 CEID50 (Egg Infection Dose) or TCID50 (Tissue Culture infection Dose) units of influenza virus. The lack of sensitivity seriously undermines the clinical usefulness of these tests.
More sensitive methods that involve the use of polymerase chain reaction (PCR) for detecting influenza virus nucleic acids have been described in a number of published articles. While these methods provide a more sensitive alternative, they generally have a number of shortcomings that make them less suitable for point-of-care use. For example, these methods generally demand complicate and lengthy sample preparation, which increases not only the cost, but also the time it takes to complete the test. These methods, particularly the so called real time PCR, also require expensive equipment and well-trained technicians, which cannot be afforded by most point-of-care facilities, e.g., the physician's offices.
In an attempt to prepare a reagent that would provide better sensitivity for influenza virus detection, a substrate for chemiluminescent influenza viral neuraminidase detection was synthesized (Analytical Biochemistry 2000; 280: 291-300). The substrate is a conjugate between N-acetyl-neuraminic acid and spiroadamantyl-1,2-dioxetane (herein referred to as dioxetane) through the 2′ position of the sialic acid. Cleavage of the linkage by neuraminidase releases the dioxetane, which oxidizes to generate chemiluminescence under alkaline conditions.
Therefore, this substrate could be used for the detection of neuraminidase activity, including that of influenza virus, as a means of detecting a pathogen such as influenza virus.
One shortcoming of the sialic acid-dioxetane substrate or methylated form of this substrate is that the pH for the neuraminidase reaction and chemiluminescence reaction is significantly different (e.g., pH 6.5 vs. pH 11). This makes it necessary to separate these two reactions, which increases the complexity and cost of the assay.
Similar problems also exist for the detection of other pathogens and diseases. Therefore, there is a need for reagents or kits that enables simple, rapid and sensitive detection of influenza virus in a clinical, animal or environmental sample.